It has recently been discovered that an interleukin-1.beta. converting enzyme (ICE) is responsible for cleaving pro-IL-1.beta. into mature and active IL-1.beta. and is also responsible for programmed cell death (or apoptosis), which is a process through which organisms get rid of unwanted cells.
Apoptosis, or programmed cell death, is a physiologic process important in the normal development and homeostasis of metazoans
In the nematode Caenorhabditis elegans, a genetic pathway of programmed cell death has been identified (Ellis, R. E., et al. Annu. Rev. Cell Biol., 7:663-698 (1991)). Two genes, ced-3 and ced-4, are essential for cells to undergo programmed cell death in C. elegans (Ellis, H. M., and Horvitz, H. R., Cell, 44:817-829 (1986)). It is becoming apparent that a class of cysteine proteases homologous to Caenorhabditis elegans Ced-3 play the role of "executioner" in the apoptotic mechanism (Martin, S. J., and Green, D. R. (1995) Cell 82, 349-352; Chinnaiyan, A. a. D., VM. (1996) Current Biology 6; Henkart, P. (1996) Immunity 4, 195-201). Recessive mutations that eliminate the function of these two genes prevent normal programmed cell death during the development of C. elegans. The known vertebrate counterpart to ced-3 protein is ICE. The overall amino acid identity between ced-3 and ICE is 28%, with a region of 115 amino acids (residues 246-360 of ced-3 and 164-278 of ICE) that shows the highest identity (43%). This region contains a conserved pentapeptide, QACRG (SEQ ID NO:10) (residues 356-360 of ced-3), which contains a cysteine known to be essential for ICE function.
The similarity between ced-3 and ICE suggests not only that ced-3 might function as a cysteine protease but also that ICE might act as a vertebrate programmed cell death gene. ced-3 and the vertebrate counterpart, ICE, control programmed cell death during embryonic development, (Gagliamini, V. et al., Science, 263:826:828 (1994).
Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that normally die during C. elegans embryogenesis (Yuan, J. Y., and Horvitz, H. R. (1990) Dev Biol 138, 33-41). While no mammalian homologs of ced-4 have been identified, ced-3 shares sequence similarity with interleukin-1b converting enzyme (ICE) (Yuan, J. et al(1993) Cell 75, 641-652), a cysteine protease involved in the processing and activation of pro-IL-1b to an active cytokine (Cerretti, D. P., et al (1992) Science 256, 97-100, Thornberry, N. A.,et al (1992) Nature 356, 768-774). Recently, numerous homologs of ICE/Ced-3 have been characterized, comprising a new gene family of cysteine proteases. To date, seven members of the ICE/Ced-3 family have been identified and include ICE (Cerretti, D. P., et al (1992) Science 256, 97-100), TX/ICH2/ICE rel-II (Munday, N. A., et al (1995) J Biol Chem 270, 15870-15876; Faucheu, C. et al. (1995) Embo J 14, 1914-1922; Kamens, J et al.(1995) J Biol Chem 270, 15250-1525612), ICE rel-III (Munday, N. A., et al (1995) J Biol Chem 270, 15870-15876), ICH1/Nedd-2 (Kumar, S., et al. (1994) Genes and Development 8, 1613-1626; Wang, L., et al. (1994) Cell 78, 739-750), Yama/CPP32/Apopain (Tewari, M., et al. (1995) Cell 81, 801-809; Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 30761-30764; Nicholson, D. Wet al. (1995) Nature 376, 37-43), Mch2 Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 30761-30764) and ICE-LAP3/Mch3/CMH-1 (Duan, H., et al. (1996) J. Biol. Chem. 271, 35013-35035; Fernandes-Alnemri, T., et al. (1995) Cancer Research 55, 6045-6052; Lippke, J. A., et al. (1996) The Journal of Biological Chemistry 271, 1825-1828). All family members share sequence homology with ICE/Ced-3 and contain an active site QACRG (SEQ ID NO:10) pentapeptide in which the cysteine residue is catalytic. Ectopic expression of these proteases in a variety of cells causes apoptosis. Phylogenetic analysis of the ICE/ced-3 gene family revealed three subfamilies (Chinnaiyan, A. a. D., VM. (1996) Current Biology 6; uan, H., et al. (1996) J. Biol. Chem. 271, 35013-35035). Yama, ICE-LAP3, and Mch2 are closely related to C.elegans Ced-3 and comprise the Ced-3 subfamily. ICE and the ICE-related genes, ICE rel II, and ICE rel III form the ICE subfamily, while ICH1 and its mouse homologue, NEDD-2 form the NEDD-2 subfamily. Based on similarities with the structural prototype interleukin-1b converting enzyme, ICE/Ced-3 family members are synthesized as zymogens that are capable of being processed to form active heterodimeric enzymes (Thomberry, N. A.,et al (1992) Nature 356, 768-774). It will be important to determine which family members are in fact activated in response to apoptotic stimuli. Previous studies have demonstrated that pro-Yama and pro-ICE-LAP3 are processed into active subunits in response to various death stimuli including engagement of Fas/APO-1 or treatment with staurosporine (Duan, H., et al. (1996) J. Biol. Chem. 271, 35013-35035; Chinnaiyan, A. M., et al. 1996) Journal of Biological Chemistry 271, 4573-4576). Further, the serine protease granzyme B, one of the major effectors of cytotoxic T cell-mediated apoptosis, was shown to directly activate Yama (but not ICE), in vitro (Quan, L. T., et al. (1996) PNAS 93, In Press; Darmon, A. J., et al. (1995) Nature 377, 446-448).
ICE mRNA has been detected in a variety of tissues, including peripheral blood monocytes, peripheral blood lymphocytes, peripheral blood neutrophils, resting and activated peripheral blood T lymphocytes, placenta, the B lymphoblastoid line CB23, and monocytic leukemia cell line THP-1 cells (Cerretti, D. P., et al., Science, 256:97-100 (1992)), indicating that ICE may have an additional substrate in addition to pro-IL-1. The substrate that ICE acts upon to cause cell death is presently unknown. One possibility is that it may be a vertebrate homolog of the C. elegans cell death gene ced-4. Alternatively, ICE might directly cause cell death by proteolytically cleaving proteins that are essential for cell viability.
The mammalian gene bcl-2, has been found to protect immune cells called lymphocytes from cell suicide. Also, crmA, a cow pox virus gene protein product inhibits ICE's protein splitting activity.
Clearly, there is a need for factors that are useful for inducing apoptosis for therapeutic purposes, for example, as an antiviral agent, an anti-tumor agent and to control embryonic development and tissue homeostasis, and the roles of such factors in dysfunction and disease. Further, there is clear a need for factors that are useful for reducing or halting apoptosis for therapeutic purposes, for example, to treat diseases caused or associated with apoptosis, such as, particularly Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, septic shock, sepsis, stroke, chronic inflammation, acute inflammation, CNS inflammation, osteoporosis, ischemia reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS, ALS, cererbellar degeneration, ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes, brain damage, aplastic anemia, male pattern baldness, and head injury damage. There is a need, therefore, for identification and characterization of such factors that are interleukin-1 beta converting enzyme apoptosis proteases, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.